Fertilization begins with the fusion of a sperm and an oocyte. Experiments conducted by scientists verified that fertilization does not happen simply as a result of insemination following ovulation, but several hours before ovulation. The aforesaid phenomenon is described by capacitation which refers to the physiological changes spermatozoa must undergo in order to have the ability to penetrate and fertilize an oocyte. The time taken to effectuate the physiological changes varies with species, namely 5 hours among rabbits, 1 hour among mice, 2˜3 hours among rats, and 5˜6 hours among human beings.
Although its discovery dates back to 1951, capacitation is not well understood in terms of molecular regulatory mechanism and signal transduction pathways. The capacitation-induced physiological changes in spermatozoa include increased membrane fluidity, cholesterol efflux, changes in the intracellular concentration of ions, hyperpolarization, alkalization, tyrosine phosphorylation, and hyperactivation. Up to now, controlling capacitation includes at least three pathways: cAMP/PKA-dependent pathway, receptor tyrosine kinase-dependent pathway, and non-receptor tyrosine kinase-dependent pathway. All the three pathways bring about tyrosine phosphorylation, thereby capacitating spermatozoa.
Known factors in capacitation include cholesterol, calcium ions, bicarbonate ions, ROS (reactive oxygen species), progesterone, GABA (gamma-aminobutyric acid), and decapacitation factor. Among the aforesaid factors, calcium ion is a critical regulator.
Calcium influx plays an important role in both capacitation and the subsequent acrosome reaction. Research finds a marked increase in the intracellular concentration of calcium ions in mammalian spermatozoa during capacitation. To effectuate capacitation, the extracellular concentration of calcium ions in spermatozoa varies with species, namely micromolar among mouse spermatozoa, and millimolar among human spermatozoa.
Research also finds that calcium ion concentration of 0.22 mM is required for capacitation of human spermatozoa, and that calcium ion concentration of at least 0.58 mM is required for the subsequent acrosome reaction and the binding of spermatozoa to the zona pellucida, indicating that at the moment when calcium influx takes place the required calcium ion concentration varies from stage to stage. Prior to capacitation, the intracellular concentration of calcium ions in spermatozoa is maintained at lower level by the intracellular calcium ion exchange systems in spermatozoa, such as voltage-dependent calcium channel, and Ca2+/ATPase, and Na+-Ca2+exchanger. The success rates of in vitro fertilization can be increased by introducing calcium ions into spermatozoa or triggering calcium ion-storing organelles of the spermatozoa to release calcium ions into sperm cytoplasm, so as to increase the intracellular calcium ion concentration during capacitation to thereby enhance intracellular signal delivery, promote the subsequent hyperactivation, and induce the subsequent acrosome reaction.
Ligand kisspeptins, which are discovered in human placenta and found to demonstrate high affinity to GPR54, is translated from KISS1 gene. The precursory product of KISS1 gene is a protein that contains 145 amino acids, and then the protein is hydrolyzed by protease to produce a peptide composed of 54 amino acids and known as kisspeptin-54. From the very beginning, kisspeptin-54 is called metastin, because its genes are believed to be capable of inhibiting the metastasis of melanomas.
In addition to kisspeptin-54, small-sized segment peptides derived from kisspeptin precursors are identified, including kisspeptin-14, kisspeptin-13, and kisspeptin-10. Identical structures known as RF-amide motif (Arg-Phe-NH2) are disposed at the C-terminal of the peptides. All the different length peptides can bind to GPR54 and manifest the same degree of affinity thereto.
In vitro fertilization involves taking oocytes and spermatozoa out of a female human body and a male human body, respectively, rinsing the oocytes and spermatozoa, culturing oocytes and spermatozoa together until the fusion of an oocyte and a spermatozoa occurs to begin the fertilization process, culturing the fertilized oocyte for 3˜6 days before putting it back to the female human body. Three to six instances of artificial insemination failure, severe endometriosis, and would-be mothers of advanced age are indications for in vitro fertilization.
The success rates of conventional in vitro fertilization are unsatisfactorily low, i.e., 30˜40%. The success rates of in vitro fertilization among mice varies with strains and stands at 10˜90%. Accordingly, it is imperative to improve in vitro fertilization and increase its success rates.